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3.A.4 The Arsenite-Antimonite (ArsAB) Efflux Family

The arsenite resistance (Ars) efflux pumps of bacteria consist either of two proteins (ArsB, the integral membrane constituent with twelve transmembrane spanners, and ArsA, the ATP-hydrolyzing, transport energizing subunit, as for the chromosomally-encoded E. coli system), or of one protein (the ArsB integral membrane protein of the plasmid-encoded Staphylococcus system). ArsA proteins have two ATP binding domains and probably arose by a tandem gene duplication event. ArsB proteins all possess twelve transmembrane spanners and may also have arisen by a tandem gene duplication event. Structurally, the Ars pumps superficially resemble ABC-type efflux pumps, but there is no significant sequence similarity between the Ars and ABC pumps. When only ArsB is present, the system operates by a pmf-dependent mechanism, and consequently belongs in TC subclass 2.A (the ArsB family; TC#2.A.45). When ArsA is also present, ATP hydrolysis drives efflux, and consequently the system belongs in TC subclass 3.A. ArsB therefore appears twice in the TC system but ArsA appears only once. These pumps actively expel both arsenite and antimonite.

In the E. coli ArsAB transporter, both ArsA and ArsB recognize and bind their anionic substrates. A model has been proposed in which ArsA alternates between two virtually exclusive conformations (Walmsley et al., 2001). In one, (ArsA1) the A1 site is closed but the A2 site is open, but in the other (ArsA2) the opposite is true. Antimonite [Sb(III)] sequesters ArsA in the ArsA1 conformation which catalyzes ATP hydrolysis at A2 to drive ArsA between conformations that have high (nucleotide-bound ArsA) and low (nucleotide-free ArsA) affinity for antimonite. It is proposed that ArsA uses this process to sequester Sb(III) and eject it into the ArsB channel (Walmsley et al., 2001). ArsD is a metallochaperone that delivers trivalent metalloids [As(III) or Sb(III)] to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Transfer from ArsD to ArsA occurs with a conformation of ArsA that transiently forms during the catalytic cycle (Yang et al., 2010).

Homologues of ArsB are found in Gram-negative and Gram-positive bacteria as well as cyanobacteria, and several paralogues are sometimes found in a single organism. Homologues are also found in archaea and eukarya. Among the distant homologues found in eukaryotes are members of the DASS family (TC #2.47) including the rat renal Na+:sulfate cotransporter (spQ07782) and the human renal Na+:dicarboxylate cotransporter (gbU26209). The ArsB proteins may therefore be members of a superfamily (called the IT (ion transporter) superfamily) that includes this DASS family (Rabus et al., 1999). However, ArsB has uniquely gained the ability to function in conjunction with ArsA in order to couple ATP hydrolysis to anion efflux. Additional distant homologues of ArsB proteins may include members of the NhaB (TC #2.A.34) and NhaC (TC #2.A.35) Na+:H+ antiporter families.

At the interface of these two halves are two nucleotide-binding domains and a metalloid-binding domain (Ruan et al., 2008). Cys-113 and Cys-422 have been shown to form a high-affinity metalloid binding site. The crystal structure of ArsA shows two other bound metalloid atoms, one liganded to Cys-172 and His-453, and the other liganded to His-148 and Ser-420. There is only a single high-affinity metalloid binding site in ArsA. Cys-172 controls the affinity of this site for metalloid and hence the efficiency of metalloactivation of the ArsAB efflux pump (Ruan et al., 2008).

Many ars operons contain only three genes, arsRBC, but five-gene ars operons have two additional genes, arsD and arsA. These two genes are usually adjacent to each other. ArsA from Escherichia coli plasmid R773 is an ATPase that is the catalytic subunit of the ArsAB As(III) extrusion pump while ArsD (AAA93060; 120aas) is an arsenic chaperone to the ArsAB pump, transferring the trivalent metalloids As(III) and Sb(III) to the ArsA subunit of the pump. This increases the affinity of ArsA for As(III), resulting in increased rates of extrusion and increased resistance to arsenite. ArsD residues: Cys12, Cys13 and Cys18 are required for delivery of As(III) to and activation of the ArsAB pump (Lin et al, 2007).

ArsA homologues are found in bacteria, archaea and eukarya (both animals and plants), but there are far fewer of them in the databases than ArsB proteins, suggesting that many ArsB homologues function by a pmf-dependent mechanism. ArsA proteins are homologous to nitrogenase iron (NifH) proteins of bacteria and protochlorophyllide reductase iron sulfur ATP-binding proteins of cyanobacteria, algae and plants.

The overall reaction catalyzed by ArsB-ArsA is:

Arsenite or Antimonite (in) + ATP ⇌ Arsenite or Antimonite (out) + ADP + Pi

This family belongs to the: ArsA ATPase (ArsA) Superfamily.

References associated with 3.A.4 family:

Baker-Austin, C., M. Dopson, M. Wexler, R.G. Sawers, A. Stemmler, B.P. Rosen, and P.L. Bond. (2007). Extreme arsenic resistance by the acidophilic archaeon 'Ferroplasma acidarmanus' Fer1. Extremophiles 11: 425-434. 17268768
Bruhn, D.F., J. Li, S. Silver, F. Roberto and B.P. Rosen (1996). The arsenical resistance operon of IncN plasmid R46. FEMS Microbiol. Lett. 139: 149-153. 8674982
Kuroda, M., S. Dey, O.I. Sanders and B.P. Rosen (1997). Alternate energy coupling of ArsB, the membrane subunit of the Ars anion-translocating ATPase. J. Biol. Chem. 272: 326-331. 8995265
Lee, S.-T., R.D. Nicholls, M.T.C. Jong, K. Fukai and R.A. Spritz (1995). Organization and sequence of the human P gene and identification of a new family of transport proteins. Genomics 26: 354-363. 7601462
Lin, Y.F., J. Yang, and B.P. Rosen. (2007). ArsD: an As(III) metallochaperone for the ArsAB As(III)-translocating ATPase. J. Bioenerg. Biomembr. 39(5-6): 453-458. 17955352
Rabus, R., D.L. Jack, D.J. Kelly and M.H. Saier, Jr. (1999). TRAP transporters: an ancient family of extracytoplasmic solute-receptor-dependent secondary active transporters. Microbiology 145: 3431-3445. 10627041
Rensing, C., M. Ghosh and B.P. Rosen (1999). Families of soft-metal-ion transporting ATPase. J. Bacteriol. 181: 5891-5897. 10498699
Rosen, B.R. (1996). Bacterial resistance to heavy metals and metalloids. JBIC 1: 273-277.
Ruan, X., H. Bhattacharjee, and B.P. Rosen. (2008). Characterization of the metalloactivation domain of an arsenite/antimonite resistance pump. Mol. Microbiol. 67: 392-402. 18067540
Silver, S., G. Ji, S. Bröer, S. Dey, D. Dou and B.P. Rosen (1993). Orphan enzyme or patriarch of a new tribe: The arsenic resistance ATPase of bacterial plasmids. Mol. Microbiol. 8: 637-642. 8332056
Soo, R.M., J. Hemp, D.H. Parks, W.W. Fischer, and P. Hugenholtz. (2017). On the origins of oxygenic photosynthesis and aerobic respiration in Cyanobacteria. Science 355: 1436-1440. 28360330
Walmsley, A.R., T. Zhou, M.I. Borges-Walmsley and B.P. Rosen (2001). A kinetic model for the action of a resistance efflux pump. J. Biol. Chem. 276: 6378-6391. 11096086
Xu, C., T. Zhou, M. Kuroda and B.P. Rosen (1998). Metalloid resistance mechanisms in prokaryotes. J. Biochem. 123: 16-23. 9504403
Yang, J., S. Rawat, T.L. Stemmler, and B.P. Rosen. (2010). Arsenic binding and transfer by the ArsD As(III) metallochaperone. Biochemistry 49: 3658-3666. 20361763